The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Summary

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Abstract

Explant culture allows manipulation of developing organs at specific time points and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.

In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel's cartilage, nasal glands, tongue, and ear.

Introduction

For a number of experiments it is important to be able to culture tissue ex vivo to follow development. Culture of developing tissue provides access at defined periods of development and allows manipulation of genes by the addition of factors to the culture medium, or on loaded beads, and by the use of transfection and electroporation¹. For many experiments it is important to be able to visualize the tissue as it grows, for example, to follow the fate of lineage labeled cells as the tissue undergoes morphogenesis. This can be particularly problematic for tissues that develop deep within the embryo, which are not obvious when a block of tissue from the embryo is cultured. Teeth are good examples of this, as they develop within the mandible, maxilla and frontal nasal process. When the whole mandible is cultured the superficial structures of the tooth can be viewed but changes in morphology can only be analyzed after sectioning of fixed tissue². We have adapted a live slice culture technique allowing us to follow tooth germ development and providing access to the different parts of the tooth during development. The technique cultures the tooth germ slices at the gas-liquid interface, using a modified Trowell method³. These slice cultures have been very useful in directly following morphogenesis of the tooth, and allowing lineage tracing of distinct components, such as the enamel knot and the dental papilla and follicle^1,4-7. The technique is not limited to mouse embryos and has successfully been used to culture live slices of pig and snake dental tissue^8,9. In addition to the benefit of being able to visualize tooth development, the slice method also has the advantage that the thin slices of tissue have increased access to nutrients from the medium and air from the incubator. This results in improved growth of the tooth germs, which match development in vivo, and show invasion of endothelial cells into the papilla⁷. In contrast, tooth germs in whole mandible cultures develop slower than those in vivo and the center of the culture is often necrotic in long-term cultures. In slice culture, the tooth develops within a slice of the jaw, and its interaction with the surrounding developing bone and other tissues can be monitored. In our method the tissue is chopped straight after dissection with no need to embed in a support medium^10,11 and no need for any system to attach the tissue to the chopping block. The method is therefore noninvasive and rapid, allowing many mandibles to be sectioned in one session.

Protocol

1. Set Up

1. Sharpen the dissection instruments (using a sharpening stone lubricated with mineral oil) and sterilize prior to use for organ culture using a 70% ethanol spray and dry-heat sterilization. Sharpen the dissection needles using electrolysis in 2 M sodium hydroxide using a 12 V supply.
2. Prepare the culture medium used for the dissection and culture of embryonic organs, consisting of Advanced Dulbecco's Modified Eagle Medium F12 (DMEM F12) supplemented with 1% GlutaMAX and 1% penicillin-streptomycin.

2. Embryo Dissection

1. Cull a timed mated pregnant female using a schedule one method as approved by the Home Office or other governing body. Dissect away the skin around the lower abdomen, and cut open the abdomen using scissors. Locate the two uterine horns, which connect together at the lower midline.
2. Dissect out the uterus and place in a tube filled with medium on ice. When on ice, the tissue can be left for several hours.
3. Remove the uterus and place in a Petri dish in medium under the microscope. Where possible embryo dissections should be performed in a laminar-flow hood to minimize contamination.
4. Dissect out the embryos from the uterus and free them from their extraembryonic tissues.
5. Decapitate the embryos using a tungsten needle and transfer the heads into a fresh dish of medium.
6. Isolate the mandibles from the heads by placing a needle into the oral cavity and cut through towards the back of the head (Figures 1A and 1B). Isolated mandibles can be temporarily stored in medium on ice while waiting for chopping.
7. For whole mandible slices stages E11.5-E15.5 are ideal for tooth germ culture. After E15.5 the developing bone in the mandible is too hard for chopping. Tooth slices can still be made but the bone (dentary and forming alveolar bone) must first be removed by dissection before chopping⁹. Remove bone carefully using tungsten needles and forceps.

3. Embryo Slicing

1. Prepare the mandible tissue slices using a standard table tissue chopper (Figure 1C). Use a fresh blade after approximately 200 mandibles, and make sure it lies flush with the mounting disc when it falls.
2. Clean the mounting disc and blade with 70% ethanol before use. Care must be taken when using the machine, as the blade is very sharp.
3. Set the chopper at a cutting distance of between 200-400 μm according to the age of the specimen, and depending on whether the whole tooth germ is required. Set the blade force on maximum.
4. Transfer a mandible to the plastic mounting disc using a pipette or forceps. Arrange the mandible on the disc making sure the orientation is still clear. The developing tongue can be used as a useful landmark for determining orientation. For molars use a frontal/transverse orientation (see plane of chopping in Figure 1B), while for incisors use a sagittal slice through the mandible to reflect the different orientation of these teeth as they grow (see plane of chopping in Figure 2A).
5. Remove the excess medium from around the tissue using filter paper in order to slightly dry the mandible on the mounting disc.
6. Immediately after drying, place the disc on the circular stainless steel table of the chopper. Turn the machine on and press start. The table traverses automatically from left to right while at the same time the chopping arm carrying the blade is raised and dropped at up to 200 strokes/min.
7. Turn off the machine once the tissue has been completely sliced. Make sure the blade arm is in the raised position and remove the disc. Do not place fingers under the blade arm or leave the machine unattended when chopping.
8. Take the mounting disc and immediately add medium to the slices on the disc in order to prevent excessive drying. Carefully dislodge the sliced tissue from the bottom of the disc using a tungsten needle and then pipette into a small Petri dish of medium (Figure 2A).
9. Separate the slices using a needle and then select the tissue slices containing the tissue of interest under a stereomicroscope (Figures 1D, 1E, and 2B). Place selected slices in fresh culture dishes with medium.

4. Slice Culture

1. Prepare central-well organ culture dishes ready for culture by adding approximately 2 ml of autoclaved water to the outer well to prevent dehydration, and by positioning a metal grid across the center of the dish (Figures 3A and 3B).
2. Prepare metal grids by cutting strips approximately 4 cm x 1.5 cm from sheets of stainless steel mesh. Use a hole punch to make a hole in the center and bend the sides to create a staggered end. The grid should fit flat across the central well, lying parallel to the bottom of the dish (Figures 3A and 3B). Autoclave the grids to sterilize before use.
3. Cut a piece of transparent polyethylene terephthalate (PET) membrane (pore size 0.4 μm) large enough to cover the hole in the grid. Place the cut membrane into the dish with the selected slice.
4. Place the slice on top of the membrane and then carefully lift the membrane out of the medium, with slice flattened in the center.
5. Place the filter on the grid over the circular hole in the center, and add approximately 1 ml of culture medium to the well, up to the level of the membrane.
6. For manipulations add proteins or small molecule inhibitors to the medium (for example⁸).
7. Photograph the slice for a record of morphology at day 0 of culture.
8. Place the dishes in a humidified atmosphere of 5% CO₂ at 37 °C in an incubator.
9. Photograph the cultures at regular intervals for on average 7 days. Change the culture medium every 48-72 hr. For photography view the slices with a light source from underneath (Figures 2C-E).

5. Lineage Tracing

If lineage tracing is required, lipophilic dyes such as DiI or DiO can be injected into the tooth germ before step 4, slice culture.

1. Pull glass capillary needles using a needle puller.
2. Break the tip of the needle using forceps and place tip down into the dye solution. Leave for a few minutes while the dye fills the tip by capillary action.
3. Place the filled needle into a holder and attach via tubing to an injector or mouth aspirator.
4. Position the tip of the needle in the culture medium touching the area of the slice due to be labeled. Apply air pressure to displace a small amount of dye out of the needle and onto the tissue.
5. Remove needle and check for labeling under fluorescence.
6. Successfully labeled slices can be transferred to filters and cultured as described in step 4.

Results

In order to follow the movement of the first molar dental follicle, 250 μm frontal slices were taken through a mandible using the method described above. The dental follicle is the layer of mesenchyme that surrounds the outer enamel epithelium (OEE) of the developing tooth, and has previously been shown to take part in the formation of tissue of the periodontium ⁶. Mandibles were dissected at E14.5, the cap stage of tooth development. In the slice the outline of the dental epithelium was clear, and the condens...

Discussion

This method of tooth culture has the advantage that access to the tooth germ is excellent, allowing accurate lineage tracing and placement of beads within the epithelium or the mesenchyme. Defined regions of the developing tooth germ can therefore be specifically targeted. During culture the changing morphology of the tooth germ can be followed, and the effect of manipulations quickly assessed.

The method, however, is only suitable for young tooth germs before substantial formation of hard tis...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethanol	VWR	101077Y	100% ethanol was diluted in distilled H2O to 70%.
DMEM F12	Gibco	12634-010	Advanced Dulbecco's Modified Eagle Medium F12
GlutaMAX	Gibco	35050-061
Penicillin-streptomycin	Sigma	P0781
DiI (Molecular probes)	Vybrant	V-22885	Cell-labeling solution
	Invitrogen		Cell tracker CM-DiI, C-7000
DiO (Molecular probes)	Vybrant	V22886
Geminator 500	Thomas	Thomas No. 3885A20	Dry-heat sterilization
McIlwain tissue chopper	Ted Pella, Inc.	10180	Standard table
Organ culture dish (Center-Well Organ Culture Dish)	Falcon	353037
Membranes (Cell Culture Inserts, 0.4 μm pore size)	BD Falcon	353090	PET track-etched membrane, 6-well format
Metal grids (Stainless Steel - AISI 304 - Mesh)	Goodfellow	FE228710	(Fe/Cr18/Ni10)
AutoFlow Direct Heat CO2 Incubator	Nuaire	NU-5500
Picospritzer III	Intracel Ltd	051-0500-900 0-100 psi	Single channel picospritzer III
Glass capillary with filament 1 mm	WPI	TW100F-4
Tungsten wire 0.1 mm	Goodfellow	W005138
Tungsten wire 0.38 mm	Goodfellow	W005155
Aspirator tubes	Sigma	A5177	Used for mouth aspiration lineage tracers